Hereditary Non-Polyposis Colorectal Cancer (HNPCC or Lynch syndrome) is an inherited form of colorectal cancer (CRC) caused by germ line mutations in DNA mismatch repair (MMR) genes. This leads to CRCs with a phenotype called microsatellite instability (MSI), and the resulting tumors have several unique clinical characteristics. Patients with HNPCC are at very high risk for CRC and cancers of other organs, often develop cancers at uncharacteristically young ages, and are at risk for multiple primary malignancies. Current challenges in HNPCC include our inability to find a germ line mutation in all families with the disease an incomplete understanding of the evolution of the disease at the cellular level, and uncertainty about how defective DNA mismatch repair mechanistically leads to tumor formation. This application proposes to develop novel methods that will extend our ability to diagnose large genomic deletions in the DNA MMR gene hMSH2, which are mediated by Alu-recombination events. Studies are proposed to determine the mechanism(s) by which the "second hit" occurs in target colorectal tissues that have developed cancer. A new model is proposed to study how inappropriate "relaxation" of DNA MMR occurs in response to oxidative stress, by measuring rates of synthesis and degradation of MMR proteins. Studies on the down regulation of DNA MMR activity have been designed to provide insight into the mechanism responsible for the enigmatic MSl-low phenotype, which is distinct from HNPCC, but may account for cancers with a form of MSI that occur in chronically inflamed mucosa. Finally, a plan is outlined to selectively inhibit the major DNA MMR genes hMSH2 and hMLH1 using siRNA technology, to determine the effects on mutation rates, cell growth, tolerance of mutational damage, and alterations in the cell cycle. This is a comprehensive research plan developed with a long-term aim of understanding how tumors develop in HNPCC, towards an overall goal of translating these insights into improved preventive strategies for patients at risk for CRC.